L-Arginine Ameliorates Defective Autophagy in GM2 Gangliosidoses by mTOR Modulation.

AIMS: Tay-Sachs and Sandhoff diseases (GM2 gangliosidosis) are autosomal recessive disorders of lysosomal function that cause progressive neurodegeneration in infants and young children. Impaired hydrolysis catalysed by β-hexosaminidase A (HexA) leads to the accumulation of GM2 ganglioside in neuronal lysosomes. Despite the storage phenotype, the role of autophagy and its regulation by mTOR has yet to be explored in the neuropathogenesis. Accordingly, we investigated the effects on autophagy and lysosomal integrity using skin fibroblasts obtained from patients with Tay-Sachs and Sandhoff diseases. RESULTS: Pathological autophagosomes with impaired autophagic flux, an abnormality confirmed by electron microscopy and biochemical studies revealing the accelerated release of mature cathepsins and HexA into the cytosol, indicating increased lysosomal permeability. GM2 fibroblasts showed diminished mTOR signalling with reduced basal mTOR activity. Accordingly, provision of a positive nutrient signal by L-arginine supplementation partially restored mTOR activity and ameliorated the cytopathological abnormalities. INNOVATION: Our data provide a novel molecular mechanism underlying GM2 gangliosidosis. Impaired autophagy caused by insufficient lysosomal function might represent a new therapeutic target for these diseases. CONCLUSIONS: We contend that the expression of autophagy/lysosome/mTOR-associated molecules may prove useful peripheral biomarkers for facile monitoring of treatment of GM2 gangliosidosis and neurodegenerative disorders that affect the lysosomal function and disrupt autophagy

A Specific Activity-Based Probe to Monitor Family GH59 Galactosylceramidase, the Enzyme Deficient in Krabbe Disease

Galactosylceramidase (GALC) is the lysosomal β-galactosidase responsible for the hydrolysis of galactosylceramide. Inherited deficiency in GALC causes Krabbe disease, a devastating neurological disorder characterized by accumulation of galactosylceramide and its deacylated counterpart, the toxic sphingoid base galactosylsphingosine (psychosine). We report the design and application of a fluorescently tagged activity-based probe (ABP) for the sensitive and specific labeling of active GALC molecules from various species. The probe consists of a β-galactopyranose-configured cyclophellitol-epoxide core, conferring specificity for GALC, equipped with a BODIPY fluorophore at C6 that allows visualization of active enzyme in cells and tissues. Detection of residual GALC in patient fibroblasts holds great promise for laboratory diagnosis of Krabbe disease. We further describe a procedure for in situ imaging of active GALC in murine brain by intra-cerebroventricular infusion of the ABP. In conclusion, this GALC-specific ABP should find broad applications in diagnosis, drug development, and evaluation of therapy for Krabbe disease

Decrease in Myelin-Associated Lipids Precedes Neuronal Loss and Glial Activation in the CNS of the Sandhoff Mouse as Determined by Metabolomics

Sandhoff disease (SD) is a lysosomal disease caused by mutations in the gene coding for the β subunit of β-hexosaminidase, leading to deficiency in the enzymes β-hexosaminidase (HEX) A and B. SD is characterised by an accumulation of gangliosides and related glycolipids, mainly in the central nervous system, and progressive neurodegeneration. The underlying cellular mechanisms leading to neurodegeneration and the contribution of inflammation in SD remain undefined. The aim of the present study was to measure global changes in metabolism over time that might reveal novel molecular pathways of disease. We used liquid chromatography-mass spectrometry and 1H Nuclear Magnetic Resonance spectroscopy to profile intact lipids and aqueous metabolites, respectively. We examined spinal cord and cerebrum from healthy and Hexb−/− mice, a mouse model of SD, at ages one, two, three and four months. We report decreased concentrations in lipids typical of the myelin sheath, galactosylceramides and plasmalogen-phosphatidylethanolamines, suggesting that reduced synthesis of myelin lipids is an early event in the development of disease pathology. Reduction in neuronal density is progressive, as demonstrated by decreased concentrations of N-acetylaspartate and amino acid neurotransmitters. Finally, microglial activation, indicated by increased amounts of myo-inositol correlates closely with the late symptomatic phases of the disease

Gene transfer corrects acute GM2 gangliosidosis--potential therapeutic contribution of perivascular enzyme flow.

The GM2 gangliosidoses are fatal lysosomal storage diseases principally affecting the brain. Absence of β-hexosaminidase A and B activities in the Sandhoff mouse causes neurological dysfunction and recapitulates the acute Tay-Sachs (TSD) and Sandhoff diseases (SD) in infants. Intracranial coinjection of recombinant adeno-associated viral vectors (rAAV), serotype 2/1, expressing human β-hexosaminidase α (HEXA) and β (HEXB) subunits into 1-month-old Sandhoff mice gave unprecedented survival to 2 years and prevented disease throughout the brain and spinal cord. Classical manifestations of disease, including spasticity-as opposed to tremor-ataxia-were resolved by localized gene transfer to the striatum or cerebellum, respectively. Abundant biosynthesis of β-hexosaminidase isozymes and their global distribution via axonal, perivascular, and cerebrospinal fluid (CSF) spaces, as well as diffusion, account for the sustained phenotypic rescue-long-term protein expression by transduced brain parenchyma, choroid plexus epithelium, and dorsal root ganglia neurons supplies the corrective enzyme. Prolonged survival permitted expression of cryptic disease in organs not accessed by intracranial vector delivery. We contend that infusion of rAAV into CSF space and intraparenchymal administration by convection-enhanced delivery at a few strategic sites will optimally treat neurodegeneration in many diseases affecting the nervous system.We gratefully acknowledge support from The National Institute of Health Research-Biomedical Research Centre, an unrestricted grant from Cambridge in America, CLIMB (Children Living with Inherited Metabolic Disorders) National Tay–Sachs and Allied Diseases Association, MRC Link Programme Award, The Paul Morgan Trust and MRC core-funding (grant code U.1052.00.005)

Decrease in Myelin-Associated Lipids Precedes Neuronal Loss and Glial Activation in the CNS of the Sandhoff Mouse as Determined by Metabolomics

Sandhoff disease (SD) is a lysosomal disease caused by mutations in the gene coding for the β subunit of β-hexosaminidase, leading to deficiency in the enzymes β-hexosaminidase (HEX) A and B. SD is characterised by an accumulation of gangliosides and related glycolipids, mainly in the central nervous system, and progressive neurodegeneration. The underlying cellular mechanisms leading to neurodegeneration and the contribution of inflammation in SD remain undefined. The aim of the present study was to measure global changes in metabolism over time that might reveal novel molecular pathways of disease. We used liquid chromatography-mass spectrometry and 1H Nuclear Magnetic Resonance spectroscopy to profile intact lipids and aqueous metabolites, respectively. We examined spinal cord and cerebrum from healthy and Hexb−/− mice, a mouse model of SD, at ages one, two, three and four months. We report decreased concentrations in lipids typical of the myelin sheath, galactosylceramides and plasmalogen-phosphatidylethanolamines, suggesting that reduced synthesis of myelin lipids is an early event in the development of disease pathology. Reduction in neuronal density is progressive, as demonstrated by decreased concentrations of N-acetylaspartate and amino acid neurotransmitters. Finally, microglial activation, indicated by increased amounts of myo-inositol correlates closely with the late symptomatic phases of the disease

Characterization of Inducible Models of Tay-Sachs and Related Disease

<div><p>Tay-Sachs and Sandhoff diseases are lethal inborn errors of acid β-N-acetylhexosaminidase activity, characterized by lysosomal storage of GM2 ganglioside and related glycoconjugates in the nervous system. The molecular events that lead to irreversible neuronal injury accompanied by gliosis are unknown; but gene transfer, when undertaken before neurological signs are manifest, effectively rescues the acute neurodegenerative illness in <i>Hexb−/−</i> (Sandhoff) mice that lack β-hexosaminidases A and B. To define determinants of therapeutic efficacy and establish a dynamic experimental platform to systematically investigate cellular pathogenesis of GM2 gangliosidosis, we generated two inducible experimental models. Reversible transgenic expression of β-hexosaminidase directed by two promoters, mouse <i>Hexb</i> and human <i>Synapsin 1</i> promoters, permitted progression of GM2 gangliosidosis in Sandhoff mice to be modified at pre-defined ages. A single auto-regulatory tetracycline-sensitive expression cassette controlled expression of transgenic <i>Hexb</i> in the brain of <i>Hexb−/−</i> mice and provided long-term rescue from the acute neuronopathic disorder, as well as the accompanying pathological storage of glycoconjugates and gliosis in most parts of the brain. Ultimately, late-onset brainstem and ventral spinal cord pathology occurred and was associated with increased tone in the limbs. Silencing transgenic <i>Hexb</i> expression in five-week-old mice induced stereotypic signs and progression of Sandhoff disease, including tremor, bradykinesia, and hind-limb paralysis. As in germline <i>Hexb−/−</i> mice, these neurodegenerative manifestations advanced rapidly, indicating that the pathogenesis and progression of GM2 gangliosidosis is not influenced by developmental events in the maturing nervous system.</p></div

Inducible expression of transgenic constructs in the brain.

<p>Relative mRNA expression was analysed in mouse forebrain using primers specific for transgenic <i>Hexb</i> (black bars) and tet-transactivator (white bars), standardized to β-actin transcript. Animals used for analysis of transgene expression were <i>Hexb+/−Hex^Tg</i> or <i>Hexb+/−SYN^Tg</i>. Panel A shows expression analysis of mice bearing the Hex construct. When no doxycycline is present, transgenic <i>Hexb</i> exceeds tet-transactivator expression. Within one day of doxycycline exposure, <i>Hexb</i> expression is almost completely repressed. When doxycycline is removed, <i>Hexb</i> expression returns within six days and is stable thereafter. In SYN transgenic animals (B), suppression of transgenic <i>Hexb</i> with doxycycline resembled the Hex line. In contrast, when doxycycline was removed, transgenic <i>Hexb</i> recovered more slowly. Bars represent mean ± SEM. n = 3 per time point except the first timepoint of each A and B where n = 4. (C) Total β-hexosaminidase activity in brain lysates was measured by MUG cleavage at timepoints post doxycycline exposure to determine how long transgenic <i>Hexb</i> protein lasted in the Sandhoff mouse brain. When <i>Hexb−/−SYN^Tg</i> animals were exposed to doxycycline, low levels of β-hexosaminidase activity could still be seen one week later, and reached its minimum by two weeks of doxycycline exposure. Data points = mean ± SEM. n = 3 animals per timepoint.</p

Expression of <i>Hexb</i> from two inducible constructs throughout the Sandhoff mouse neuraxis.

<p>(A) Expression of transgenic <i>Hexb</i> from a single autoregulatory cassette was driven by either the mouse <i>Hexb</i> promoter (Hex line) or the human <i>SYN1</i> promoter (SYN line). Tet-transactivator expressed from tTA2^s coding sequence promoted expression of <i>Hexb</i> cDNA from tet-response elements (TRE). This was inhibited by doxycycline. (B) Expression of transgenic <i>Hexb</i> in different tissues was assessed with the MUG assay. β-hexosaminidase activity was found in the brain for both Hex and SYN lines, but also in the skin and skeletal muscle for the transgenic Hex mouse line. Bars = mean ± SEM. n = 3. Panel C shows expression of β-hexosaminidase activity in <i>Hexb−/−Hex^Tg</i> and <i>Hexb−/−SYN^Tg</i> mice assessed by staining using the enzyme substrate naphthol AS-BI N-acetyl-β-glucosaminide (red staining). Strong expression of β-hexosaminidase activity is seen in the cortex (C i–vi) and the cerebellum (C vii–ix) of both lines. Weaker expression is seen in the diencephalon in the SYN line (C iv–v) while activity is very weak to absent in the mid (C vi–vii) and hindbrain (C vii–ix). (D) β-hexosaminidase activity staining was associated with neurons, shown by co-labelling with NeuN (green fluorescence) in the piriform cortex (i and ii), CA1 field of Ammon's horn (iii and iv), the cerebellar cortex (v and vi) and the dorsal root ganglia (vii and viii), where only some neurons expressed transgenic β-hexosaminidase activity. Images represent staining from <i>Hexb−/−Hex^Tg</i> mice (DRG and cerebellar cortex) and <i>Hexb−/−SYN^Tg</i> mice (CA1 field and piriform cortex). ML = molecular layer, PyL = pyramidal layer, CA1 = CA1 field, PL = Purkinje neuron layer, GCL = granule cell layer. Scale bar = 50 µm.</p

Inducible transgenic constructs rescue mice from Sandhoff disease.

<p>(A) <i>Hexb−/−</i> mice that carry the Hex or SYN transgenic cassettes show an average survival of 373 or 404 days, respectively. This is a three-fold increase on <i>Hexb−/−</i> mice that do not carry a transgenic expression construct and only survive to an average of 127 days. Plots show data points overlaid with the mean ± SEM. (B) Motor performance of transgenic animals was assessed using the inverted screen test and performance measured by multiplying latency to fall (seconds) by number of hindlimb movements. <i>Hexb−/−</i> Sandhoff mice rapidly deteriorated after 14 weeks of age (green triangles). <i>Hexb−/−Hex^Tg</i> and <i>Hexb−/−SYN^Tg</i> mice showed motor performance comparable with <i>Hexb+/−</i> mice up until six months of age, by which point transgenic mice began progressive deterioration that culminated in humane endpoint at approximately one year of age. n = 6, 8, 11 and 9 mice for <i>Hexb−/−Hex^Tg</i>, <i>Hexb−/−SYN^Tg</i>, <i>Hexb−/−</i> and <i>Hexb</i>+/− respectively. Data points represent mean ± SEM.</p

Suppression of <i>Hexb</i> expression results in development of stereotypic Sandhoff disease.

<p>A to D show motor performance measured by the inverted screen test. (A) No difference exists between two groups of healthy control mice (<i>Hexb+/−</i>); with (red, n = 5) and without (blue, n = 6) doxycycline treatment starting at five weeks of age. To determine if doxycycline itself modified Sandhoff disease (B), <i>Hexb−/−</i> mice were also maintained with (red, n = 6) and without (blue, n = 10) doxycycline. (C) <i>Hexb−/−Hex^Tg</i> mice maintained steady performance on the inverted screen test (blue, n = 6). In contrast, when <i>Hexb−/−Hex^Tg</i> mice were exposed to doxycycline from five weeks of age, their performance began to deteriorate from about 20 weeks of age onward (red, n = 8). This rapid deterioration in performance mimics that of <i>Hexb−/−</i> mice (green, n = 10). (D) Similar results were obtained for <i>Hexb−/−SYN^Tg</i> mice with (red, n = 8) and without (blue, n = 8) doxycycline. Data points represent mean ± SEM. E shows survival of <i>Hexb−/−Hex^Tg</i> and <i>Hexb−/−SYN^Tg</i> mice exposed to doxycycline from five weeks of age (mean = 172.5 days, n = 8, and 175 days, n = 8, respectively). Survival of germline <i>Hexb−/−</i> mice is on average 127 days of age (n = 11), similar to the length of time inducible mice survive under doxycycline mediated suppression of transgenic <i>Hexb</i>. Plots show data points overlaid with the mean ± SEM.</p